ABSTRACT Elucidating the Molecular Mechanism of Optineurin in Glaucomatous Neurodegeneration Normal tension glaucoma (NTG) is characterized as retinal ganglion cell (RGC) degeneration in the absence of high intraocular pressure, and currently has no sight-saving therapies. Mutations in the optineurin (OPTN) gene have been associated with familial and sporadic NTG. OPTN acts as an adaptor to recruit ubiquitinated cargo to autophagosomes for clearance. OPTN also has binding domains for motor proteins and is known to play a pivotal role in cellular trafficking along the cytoskeleton. We hypothesize that a lack of cellular trafficking by glaucoma-associated mutants of OPTN in RGCs leads to a loss-of-function of OPTN to promote NTG. No studies have ever isolated the role of OPTN in RGCs in vivo. Using the mSncg promoter in AAV2-mediated RGC targeting, we will overexpress OPTN-WT or OPTN-E50K in mouse RGCs. We will also use AAV2-mSncg-Cre in floxed OPTN mice as a model of loss-of-function of OPTN. We will use in vivo retinal function assays and post- sacrifice tissue quantification and staining to define the levels of RGC degeneration and autophagy function. This will determine whether a gain- or loss-of-function of OPTN is associated with RGC degeneration. We have shown that OPTN truncation in RGCs leads to RGC degeneration at 8 weeks by in vivo assays and RGC soma and axon quantification, providing evidence for the need to study OPTN in RGCs. We have also found that after overexpression of OPTN-WT and OPTN-E50K specifically in RGCs, only OPTN-WT is expressed in the retinal nerve fiber layer, suggesting that OPTN-E50K can no longer function as an axon transport-related protein. RGCs have long projection axons through the optic nerve and must have well-functioning cellular trafficking capacity to regulate RGC health. Using immunostaining, proteomics, and axon targeting, we will determine the role of OPTN in RGC axons and the pathology that an E50K mutation incurs. In summary, our studies will reveal the role of OPTN in RGCs and uncover molecular mechanisms of OPTN-E50K-induced RGC degeneration.